Step B: coupling said epothilone intermediate and an aromatic stannane by means of a Stille coupling reaction for producing the epothilone compound, said epothilone compound being represented by the following structure: ##STR18##

and the aromatic stannane being a compound represented as (R.sub.y).sub.3 Sn--R.sub.10 wherein

R.sub.y is either n-butyl or methyl; R.sub.10 is a radical selected from a group consisting of one of the following structures: ##STR19##

7. A process for synthesizing an epothilone compound according to claim 6 wherein R.sub.10 is represented by the following structure: ##STR20##

8. A process for synthesizing an epothilone compound according to claim 6 wherein R.sub.10 is represented by the following structure: ##STR21##

9. A process for synthesizing an epothilone compound according to claim 6 wherein R.sub.10 is represented by the following structure: ##STR22##

10. A process for synthesizing an epothilone compound according to claim 6 wherein R.sub.10 is represented by the following structure: ##STR23##

Description:

TECHNICAL FIELD OF THE INVENTION

The present invention relates to epothilone analogs having side chain modifications and to methods for producing such compounds using solid phase and solution phase chemistries.

BACKGROUND OF THE INVENTION

The epothilones (1-5, FIG. 1) are natural substances which exhibit cytotoxicity against taxol-resistant tumor cells by promoting the polymerization of .alpha.- and .beta.-tubulin sub-units and stabilizing the resulting microtubule assemblies. Epothilones displace Taxol.TM. from its microtubul binding site and are reported to be about 2000-5000 times more potent than Taxol with respect to the stabilization of microtubules.

What is needed are analogs of epothilone A and B and libraries of analogs of epothilone A and B that exhibit superior pharmacological properties in the area of microtubule stabilizing agents.

Furthermore, what is needed are methods for producing synthetic epothilone A, epothilone B, analogs of epothilone A and B, and libraries of epothilone analogs, including epothilone analogs possessing both optimum levels of microtubule stabilizingeffects and cytotoxicity.

BRIEF SUMMARY OF THE INVENTION

One aspect of the invention is directed to an epothilone analog represented by the following structure: ##STR1##

In the above structure, R.sub.2 is absent or oxygen; "a" can be either a single or double bond; "b" can be either absent or a single bond; and "c" can be either absent or a single bond. However, the following provisos apply: if R.sub.2 isoxygen, then "b" and "c" are both a single bonds and "a" is a single bond; if R.sub.2 is absent, then "b" and "c" are absent and "a" is a double bond; and if "a" is a double bond, then R.sub.2, "b", and "c" are absent. R.sub.3 is a radical selected fromthe group consisting of hydrogen, methyl, --CHO, --COOH, --CO.sub.2 Me, --CO.sub.2 (tert-butyl), --CO.sub.2 (iso-propyl), --CO.sub.2 (phenyl), --CO.sub.2 (benzyl), --CONH(furfuryl), --CO.sub.2 (N-benzo-(2R,3S)-3-phenylisoserine), --CONH(methyl).sub.2,--CONH(ethyl).sub.2, --CONH(benzyl), --CH.dbd.CH.sub.2, --C.ident.CH, and --CH.sub.2 R.sub.11, wherein R.sub.11 is a radical selected from the group consisting of --OH, --O-Trityl, --O--(C.sub.1 -C.sub.6 alkyl), --(C.sub.1 -C.sub.6 alkyl), --O-benzyl,--O-allyl, --O--COCH.sub.3, --O--COCH.sub.2 Cl, --O--COCH.sub.2 CH.sub.3, --O--COCF.sub.3, --O--COCH(CH.sub.3).sub.2, --O--COC(CH.sub.3).sub.3, --O--CO(cyclopropane), --OCO(cyclohexane), --O--COCH.dbd.CH.sub.2, --0--CO--Phenyl, --O-(2-furoyl),--O-(N-benzo-(2R,3S)-3-phenylisoserine), --O-cinnamoyl, --O-(acetyl-phenyl), --O-(2-thiophenesulfonyl), --S--(C.sub.1 -C.sub.6 alkyl), --SH, --S-Phenyl, --S-Benzyl, --S-furfuryl, --NH.sub.2, --N.sub.3, --NHCOCH.sub.3, --NHCOCH.sub.2 Cl, --NHCOCH.sub.2CH.sub.3, --NHCOCF.sub.3, --NHCOCH(CH.sub.3).sub.2, --NHCOC(CH.sub.3).sub.3, --NHCO(cyclopropane), --NHCO(cyclohexane), --NHCOCH.dbd.CH.sub.2, --NHCO-Phenyl, --NH(2-furoyl), --NH-(N-benzo-(2R,3S)-3-phenylisoserine), --NH-(cinnamoyl),--NH-(acetyl-phenyl), --NH-(2-thiophenesulfonyl), --F, --Cl, I, and --CH.sub.2 CO.sub.2 H. R.sub.4 and R.sub.5 are each independently selected from hydrogen, methyl or a protecting group. R.sub.1 is a radical selected from the following structures:##STR2## ##STR3##

In a preferred embodiment, R.sub.3 is hydrogen or --CH.sub.2 R.sub.11, R.sub.11 is a radical selected from the group consisting of --OH and --F, and R.sub.1 is a radical selected from the following structures: ##STR4##

Another aspect of the invention is directed to a process for synthesizing an epothlone analog, or a salt thereof The process includes a coupling step wherein an epothilone intermediate and an aromatic stannane are coupled by means of a Stillecoupling reaction for producing the epothilone analog. The epothilone is represented by the following structure: ##STR5##

The epothilone intermediate is represented by the following structure: ##STR6##

FIG. 16 illustrates the general route to synthesis various side-chain modified epothilone B analogs having pyridine and imidazole modifications. Some of the conditions in the figure are published as follows: Nicolaou et al. Tetrahedron, 1998,54, 7127-7166.

The invention is directed to epothilone analogs and methods for producing such analogs based on approaches used to synthesize epothilones A and B. One aspect of the invention relates to the use of an improved Stille coupling strategy to completea total synthesis of epothilone E from vinyl iodide 7 and thiazole-stannane 8h. The central core fragment 7 and its trans-isomer 11 were prepared from triene 15 using ring-closing metathesis (RCM), and were subsequently coupled to a variety ofalternative stannanes to provide a library of epothilone analogs 18a-o and 19a-o. The Stille coupling approach was then used to prepare epothilone B analogs from the key macrolactone intermediate 24 which was, itself, synthesized by a macrolactonizationbased strategy.

Another aspect of the invention is directed to the chemical synthesis of the C12,13-cyclopropyl analogs of the epothilones. These and several other epothilone analogs have been synthesized and then screened for their ability to induce tubulinpolymerization and death of a number of tumor cells as described below.

The following examples illustrate methods for the total synthesis of the epithilone analogs. The examples represent exemplary conditions which demonstrate the versatility of the methodology and are not meant to be restrictive with the modelsdisclosed.

EXAMPLE 1

Total Synthesis of Epothilone E and Related Side-Chain Modified Analogs via a Stille Coupling Based Strategy

With the necessary components in hand, the critical Stille couplings could now be investigated. In the event, two alternative sets of reaction conditions proved adequate (FIG. 3). Procedure A involved heating a toluene solution of the desiredvinyl iodide (7 or 11) with the appropriate stannane 8 in the presence of catalytic amounts of Pd(PPh.sub.3).sub.4 at 80-100.degree. C. for between 15 and 40 min. This protocol was used to couple stannanes 8a-c, 8e-i and 8n. The remaining stannanes,8d, 8j-m and 8o (epothilones 18o and 19o, the products of coupling of stannane 8o with vinyl iodides 7 and 11, respectively, were isolated as C17-methyl ketones and not ethyl enol ethers) were coupled using an alternative, milder method, procedure B, inwhich a mixture of vinyl iodide (7 or 11) and stannane 8 in DMF was treated with PdCl.sub.2 (MeCN).sub.2 at 25.degree. C.

The coupling of vinyl iodide 7 and stannane 8h provided macrolactone 18h which served as the precursor to the natural epothilone E (3) (FIG. 6a). The total synthesis was completed by epoxidation with in situ generated methylperoxycarboximidicacid (H.sub.2 O.sub.2, KHCO.sub.3, MeCN, MeOH, 25.degree. C.; Chaudhuri et al. J. J. Org. Chem. 1982, 47, 5196-5198) furnishing epothilone E (3) (66% based on 50% conversion), which exhibited identical physical characteristics (.sup.1 H and .sup.13 CNMR, [.alpha.].sub.D) to those published in the art.

At this stage, we postulated that the Stille coupling approach could be extended to provide facile access to a variety of side-chain modified analogs of epothilone B (2). The impetus for this development was two-fold. Firstly, epothilone B isthe most active of the epothilones and, therefore, warranted further investigation. Secondly, the C26 position of this compound has proved to be a fertile site for modification, and it was felt that analogs possessing a combination of these twovariables could be interesting for further biological evaluation. The retrosynthetic analysis of epothilone analogs possessing these dual modifications is shown in FIG. 6b and requires the preparation of the crucial vinyl iodide core fragment 24. Amacrolactonization strategy similar to that used in our synthesis of epothilone B and a variety of epothilone analogs was thought to be most suitable for this task.

With intermediate 24 in hand, the Stille coupling protocol could then be employed to attach the desired heterocyclic moiety. The mild procedure B, employing PdCl.sub.2 (MeCN).sub.2 was thought to be the most practical and efficient process andwas utilized in the preparation of C26 hydroxy epothilones 45-48 (FIG. 9) from the vinyl iodide 24 and the appropriate stannanes 8 (see FIGS. 4 and 5). Unfortunately, these conditionswere not suitable for the coupling of 24 and vinyl stannane 8q (seeFIG. 5). Recourse to the alternative procedure A provided access to the desired epothilone 49, albeit, in poor yield.

The presence of the C26 hydroxy functionality provided a convenient handle for further elaboration of the epothilone products. For example, the C26 alcohols 45-47 and 49 were treated with DAST (CH.sub.2 Cl.sub.2, -78.degree. C.) to furnishfluorinated epothilone analogs 50-53 in moderate yields as shown in FIG. 9. Alternatively, asymmetric epoxidation of substrates 45 and 46 under Katsuki-Sharpless conditions [(+)-DET, Ti(i-PrO).sub.4, t-BuOOH, 4 .ANG. molecular sieves, CH.sub.2Cl.sub.2, -40.degree. C.; Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1980, 102, 5976-5978] afforded epothilones 54 and 55, respectively. Subsequent treatment with DAST (CH.sub.2 Cl.sub.2, -78.degree. C.) provided additional analogs 58 and 59,again in moderate yield. At this juncture, a more efficient approach to epoxides such as 54 and 55 was envisaged in which asymmetric epoxidation of vinyl iodide 24 could be achieved to give a common intermediate, which could then serve as a substratefor the Stille coupling. Despite initial reservations concerning the compatibility of the epoxide functionality with the Stille conditions, the epoxide 57 required for this approach was prepared from olefin 24 in 81% yield as described for the synthesisof 45 and 46. To our pleasant surprise, application of the standard coupling procedure B, using stannane 8r, resulted in the successful preparation of epothilone analog 56 (73% yield based on 70% conversion).

The chemistry described in this example relies on a Stille coupling approach to construct a series of epothilone analogs with diversity at the side-chain or at both the side-chain and C26 site from a common macrocyclic intermediate.

EXAMPLE 2

Synthesis and Biological Properties of C12,13-Cylopropyl-Epothilone A and Related Epothilones: Biological Evalation of Epothilone Candidates

In this example, we disclose the biological properties of a series of new epothilone analogs, whose synthesis is described elsewhere (Nicolaou et al. Angew. Chem. Int. Ed. Engl., 37, 84-87). In addition, we describe the chemical synthesis ofthe C12,13-cyclopropyl analog of epothilone A and its C12,13-trans-isomer and their biological evaluation in tubulin polymerization and certain cytotoxicity assays. The chemical synthesis of the C12,13-cyclopropyl analog of epothilone A and itsC12,13-trans-diastereoisomer has been accomplished. These and several other epothilone analogs have been screened for their ability to induce tubulin polymerization and death of a number of tumor cells. Several interesting structure-activity trendswithin this family of compounds were identified.

The results of the biological tests conducted in this study have demonstrated that, while a number of positions on the epothilone skeleton are amenable to modification without significant loss of biological activity, the replacement of theepoxide moiety of epothilone A with a cyclopropyl group leads to total loss of activity.

The synthesis of the cyclopropane analogs 300 and 400 (FIG. 11) required some rather unusual chemistry. A wide range of methods have been described in the literature for the transformation of allylic alcohols to the corresponding cyclopropylsystems, several in either diastereo- or enantioselective fashion (Kasdorf et al. Chemtracts-Organic Chemistry, 533-535). However, initial efforts employing either these methods or the classic Simmons-Smith procedure proved disappointing when attemptedon the previously prepared (Nicolaou (1997) et al. Chem. Commun. 2343-2344). macrocyclic substrate 500 (FIG. 12).

In the light of these discouraging results, a new approach was devised. Previous studies (Isono et al. (1996) J. Org. Chem., 61, 7867-7872; Hanessian et al. (1996) Tetrahedron Lett., 37, 8971-8974) have shown that cyclopropanes may be preparedfrom .gamma.-hydroxypropyl stannanes by elimination of the hydroxyl and stannyl moieties. We therefore, envisaged that if we could prepare the g-hydroxypropyl stannane systems 1000 and 1100 (FIG. 12) then alcohol derivatization and subsequentacid-catalyzed formation of a carbocation could trigger spontaneous cyclization to the required cyclopropanes 1200 and 1300 respectively (FIG. 12). It was further anticipated that the required stannanes could be prepared from allylic alcohol 900, whichin turn would be derived from the macrocylic epoxide system 600 (FIG. 12).

Thus, as shown in FIG. 12, subjecting allylic alcohol 500 (Nicolaou et al. (1997) Chem. Commun. 2343-2344) to Katsuki-Sharpless epoxidation conditions (Katsuki et al. J. Am. Chem. Soc., 102, 5974-5976) provided epoxy alcohol 600 in 92% yieldand as a single diastereoisomer (as judged by .sup.1 H NMR analysis). Tosylation of the primary alcohol also proceeded smoothly to afford tosylate 700. Subsequent treatment of 700 with sodium iodide in acetone gave the iodide 800 which, upon in situtreatment with triphenylphosphine and a catalytic amount of iodine, rapidly rearranged to allylic alcohol 900 (89% over three steps). The latter compound (900) was then exposed to tri-n-butyltin hydride in the presence of catalytic amounts ofPd(OH).sub.2 to afford the stannanes 1000 and 1100 (96% yield based on ca. 52% conversion) albeit, with modest diastereoselectivity (1000:1100; ca. 2:1). It was expected that, while elaboration of the C12-(R)-diastereoisomer 1000 would lead to thecis-cyclopropane 1200, the isomeric stannane 1100 could permit access to the equally interesting C12,13-trans-cyclopropane system 1300. Thus, treatment of 1000 with thionyl chloride and pyridine in dichloromethane at -78.degree. C., followed by warmingto room temperature over five hours, promoted the required elimination, leading to an inseparable mixture of 1200 and elimination product 1400. Desilylation (HF.pyr./THF) then allowed separation of the two components, providingC12,13-cis-cyclopropyl-epothilone A (300) (20% yield for two steps) and elimination product 1500 (62% yield for two steps)--FIG. 12.

In an analogous fashion, stannane 1100 was efficiently converted to cyclopropane system 400. Thus, following mesylation of the secondary hydroxyl group in 1100, exposure to silica gel facilitated ring closure, generating 1300 in excellent yield(89%). Finally, desilylation as before (HF.pyr./THF) afforded C12,13-trans-cyclopropyl-epqthilone A (400) in 90% yield. In both cases (300 and 400) the stereochemistry of the cyclopropane moiety was established by detailed 1H NMR experiments(1H-1H-COSY and NOESY).

The tubulin assembly and cytotoxicity data against certain tumor cell lines of cyclopropyl analogs 300 and 400, together with those of a number of other epothilone analogs recently prepared in these laboratories (Nicolaou et al. (1998) Angew. Chem. Int. Ed. Engl., 37, 84-87) are shown in FIGS. 13-14. Examination of entries 1 and 2 clearly shows that replacement of the epoxide moiety with a cyclopropane system has a profound effect on both the tubulin polymerization and cytotoxic propertiesof the molecules. In order to more fully comprehend this drastic reduction in potency, we resorted to computational chemistry techniques to examine the conformations of 300 as compared to the parent epothilone A (1). We suspected that the partial sp2character of the "banana bonds" of the cyclopropyl ring was possibly leading to distortion of the normal conformation of the epothilone framework, thereby preventing the molecule from adopting the required shape for binding to tubulin.

As shown in FIGS. 13-14, the substitution of an epoxide for a cylopropane moiety does indeed cause rather drastic changes to the minimum-energy conformation of epothilone A (1). The significant differences in the 1H NMR spectra of compounds 1and 300 were also in support of the drastic conformational changes imposed on the epothilone A skeleton by the cyclopropane ring. Similarly, the C12,13-trans-cyclopropyl-epothilone analog 400 was found to be devoid of any tubulin polymerization andcytotoxicity properties as compared to its epoxide counterpart (1600) and epothilone A (1) itself (see FIG. 13, entries 1-4).

A number of additional trends are apparent from examination of the remaining data in FIG. 13. Although analogs without the epoxide moiety showed tubulin binding activity, for the most part they displayed very low levels of cytotoxic activityagainst the tumor cell lines examined. The trends discussed below, therefore, are based on levels of tubulin polymerization. As expected, epothilone B type analogs (entries 36-52) generally exhibited higher levels of activity than those of epothilone A(1, entry 1) and related analogs (entries 5-35). In comparing non-epoxidized substrates (entries 6-35), the C12,13-cis systems generally showed higher levels of tubulin polymerization than the corresponding C12,13-trans systems (compare entries 9-13with 24-28).

Some more specific trends also became evident on comparing the C12,13-cis-olefins (entries 6-20). The presence and position of the nitrogen atom in the side chain heterocycle seems to be important. Compound 2600 (entry 15), in which thenitrogen atom is in its normal position adjacent to C18, but the sulfur of the thiazole has been relocated, still displayed good activity. However, compound 2500 (entry 14) where the nitrogen has been moved, was inactive. These trends were mirrored inthe cases of the C12,13-trans-olefins (entries 29 and 30). A similar effect can be seen with the pyridine analogs 3000 and 4500 (entries 19 and 34). Previously synthesized pyridine-containing analogs in which the nitrogen atom was adjacent to C18displayed good levels of activity (Nicolaou (1997) et al. Angew. Chem. Int. Ed. Engl., 36, 2097-2102), whereas 3000 and 4500 showed low levels of tubulin polymerization. Clearly, altering the position of the nitrogen by one atom, has severeimplications on activity.

Entries 16-17 and 31-32, where the thiazole of the epothilones had been replaced by either a furan or thiophene system, demonstrate that complete removal of the nitrogen leads to a considerable loss of tubulin polymerization activity. Substitution of the five-membered heterocycle with a six-membered carbocyclic moiety (entries 18 and 33), resulted in analogs with low activity. As can be seen from entries 20 and 35, removal of the heterocycle altogether resulted in essentiallycomplete loss of activity.

Modification at C22 (entries 6-13) seems well tolerated, provided the substituent is not too sterically demanding. For example, hydroxymethylene (1700), fluoromethylene (1900) and thiomethylether (2200) compounds showed reasonable activity,whereas the larger acetate (1800), ethoxythiazole (2100), long-chain acetate (2300) and piperidine (2400) derivatives, were somewhat less active. A similar trend was seen in the C12,13-trans systems (entries 21-28). Alteration at C26 (entries 36-52)seemed to be fairly well tolerated with high levels of activity being displayed by the fluoromethylene-olefins 5200-5500, fluoromethylene-epoxides 5900 and 6000, and the ethyl-epoxides 6100-6300. However, the C26- hydroxy olefins 4600-5100 andC26-hydroxy epoxides 5600-5800 were somewhat less active.

The success of taxol as a therapeutic agent epitomizes the value of tubulin-polymerization-microtubule stabilizing agents in the fight against cancer. The similar mode of action and improved potency of the epothilones, particularly againsttaxol-resistant tumor cell lines, has made them of particular importance as potential anti-cancer drugs, especially in cases where taxol fails. A fuller understanding of the structural requirements of the epothilones for biological activity shouldfacilitate their further development as potential anticancer agents.

In this study, the biological activities of a structurally diverse set of modified epothilones have been investigated and several useful trends noted. The biological action of the epothilones seems particularly sensitive to the location of basicheteroatoms in the side chain, and to the relative steric bulk of side-chain substituents. Furthermore, additional alterations at C26 may be tolerated resulting in analogs possessing varying degrees of activity. An important conclusion from this workwas the finding that substitution of the epoxide moiety of epothilone A (1) by a cyclopropyl group leads to total loss of activity, presumably due to drastic conformational changes imposed by this substitution.

EXAMPLE 3

Use of Additional Stannanes to Synthesize Side Chain Modified Epothilone Analogs as Illustrated in FIGS. 16 and 19

Use of the Stille coupling procedure to prepare a number of side chain modified epothilone analogs from the common precursors 57, and 800 is described in FIGS. 16 and 19. Synthesis of vinyl iodide 7002 was achieved from the previously reportedC26-hydroxy compound and involved conversion to diiodide 7001 and subsequent reduction using NaBH.sub.3 CN. Coupling to the epothilone E side chain has been achieved and the coupling of a number of pyridines and imidazoles is accomplished via couplingof numerous alternative side chains with the aromatic stannanes as shown in FIG. 16 and 19 using the standard methods outlined herein.

EXAMPLE 4

Synthesis of C-17 Desmethylepothilone B as Illustrated in FIGS. 17-18

The synthesis of C17 desmethylepothilone B is described in FIGS. 17-18 using standard methods outlined herein. The synthetic route utilizes a macrolactonization reaction to prepare the core of the molecule and the strategy closely parallels thatused in previous syntheses of epothilones A and B and related analogs. A new feature of the route is the removal of the C26 hydroxy substituent which is itself used to control the C12,13 Sharpless epoxidation reaction. Removal of this alcoholfunctionality is achieved by its conversion to the iodide and subsequent reduction using NaBH.sub.3 CN. The target compound is of interest since without the C17 methyl substituent the conformation of the thiazole side-chain will be altered and it isanticipated that this will place the important basic nitrogen of the thiazole in a preferential orientation (FIGS. 17-18).

Representative Procedures for Stannane Synthesis these Procedures are General and can be Used to Synthesize all Stannanes as shown in FIG. 19: All Reagents are Commercially Available from Aldrich, Sigma, Fluka or are Well Known in the Art

General Procedure A: A solution of bromothiazole (1.0 equiv) in degassed toluene (0.1 M), was treated with hexamethylditin (10 equiv) and Pd(PPh.sub.3).sub.4 (0.1 equiv) and the mixture was heated at 100.degree. C. for 3 h. The reaction mixturewas cooled to 25.degree. C. and purified by flash column chromatography (silica gel; pre-treated with Et.sub.3 N, 50% ether in hexanes) to afford the desired stannane (93%)

General Procedure B: To a solution of bromothiazole (1.0 equiv) in ether (0.07M) at -78.degree. C., was added n-BuLi (1.2 equiv) and the resulting mixture was stirred at -78.degree. C. for 10 min. Tri-n-butyltin chloride (1.2 equiv) was thenadded, the solution stirred at -78.degree. C. for 10 min, and then slowly warmed to 25.degree. C. over a period of 1 h. The reaction mixture was diluted with hexane and passed through silica gel eluting with 20% EtOAc in hexanes. Flash columnchromatography (silica gel; pre-treated with Et.sub.3 N, 5% ether in hexanes) furnished the desired stannane (85%).

Synthetic Protocals

General: All reactions were carried out under an argon atmosphere with dry, freshly distilled solvents under anhydrous conditions, unless otherwise noted. Tetrahydrofuran (THF) and diethyl ether (ether) were distilled from sodium-benzophenone,and dichloromethane (CH.sub.2 Cl.sub.2), benzene (PhH), and toluene from calcium hydride. Anhydrous solvents were also obtained by passing them through commercially available activated alumina columns. Yields refer to chromatographically andspectroscopically (.sup.1 H NMR) homogeneous materials, unless otherwise stated. All solutions used in workup procedures were saturated unless otherwise noted. All reagents were purchased at highest commercial quality and used without furtherpurification unless otherwise stated.

2-Hydroxymethyl-4-bromothiazole 21h as illustrated in FIG. 4. To a solution of 2,4-dibromothiazole 20 (50 mg, 0.206 mmol, 1.0 equiv) in anhydrous ether (2.0 mL, 0.1 M) at -78.degree. C., was added n-BuLi (154 mL, 1.6 M in hexanes, 0.247 mmol,1.2 equiv), and the resulting solution was stirred at the same temperature for 30 min. DMF (32 mL, 0.412 mmol, 2.0 equiv) was then added at -78.degree. C. and, after being stirred at -78.degree. C. for 30 min, the reaction mixture was slowly warmed upto 25.degree. C. over a period of 2 h. Hexane (2.0 mL) was added and the resulting mixture was passed through a short silica gel cake eluting with 30% EtOAc in hexanes. The solvents were evaporated to give the crude aldehyde 22 (50 mg), which was useddirectly in the next step.

Tubulin polymerization was determined by the filtration-colorimetric method, developed by Bollag et Cancer Res. 1995, 55, 2325-2333. Purified tubulin (1 mg/mL) was incubated at 37.degree. C. for 30 minutes in the presence of each compound (20mM) in MEM buffer [(100 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.75, 1 mM ethylene glycol bis(b-aminoethyl ether), N,N,N',N'-tetraacetic acid, and 1 mM MgCl.sub.2 ]; the mixture was then filtered to remove unpolymerized tubulin by using a 96-wellMillipore Multiscreen Durapore hydrophillic 0.22 mm pore size filtration plate; the collected polymerized tubulin was stained with amido black solution and quantified by measuring absorbance of the dyed solution on a Molecular Devices Microplate Reader. The growth of all cell lines was evaluated by quantitation of the protein in 96-well plates as described previously. Briefly, 500 cells were seeded in each well of the plates and incubated with the various concentrations of the epothilones at 37.degree. C. in a humidified 5% CO2 atmosphere for four days. After cell fixation with 50% trichloroacetic acid, the optical density corresponding to the quantity of proteins was measured in 25 mM NaOH solution (50% methanol: 50% water) at a wavelength of 564 nm. The IC50 was defined as the dose of drug required to inhibit cell growth by 50%.

FIGS. 16-18 are shown using conditions described in Nicolaou et al. J. Am. Chem. Soc.,1997, 119, 7974-7991 and those as indicated in the description of figures above.

Vinyl iodide 7002 as illustrated in FIG. 16. Diiodide 7001 (1 equiv.; from 57) and sodium cyanoborohydride (10 equiv.) were dissolved in anhydrous HMPA (0.2 M) and the resulting mixture heated at 45-50.degree. C. for 48 h. After cooling to roomtemperature, water was added and the aqueous phase extracted four times with ethyl acetate. The combined organic fractions were dried (Na.sub.2 SO.sub.4) and passed through a short plug of silica gel to remove traces of HMPA (eluting with 50% ethylacetate in hexanes). Following evaporation of solvents, the residue was purified by preparative thin layer chromatography (eluting with 50% ethyl acetate in hexanes) to provide pure vinyl iodide 7002 (84%).

Ylide 708 as illustrated in FIG. 17. Phosphonium salt 7007 (1.0 equiv.) was dissolved in THF (0.2 M) and the solution was cooled to 0.degree. C. Potassium hexamethyldisilylamide (KHMDS, 2.0 equiv.) was slowly added and the resulting mixture wasstirred for 30 min. The mixture was then cooled to -78.degree. C. and methyl chloroformate was added dropwise. Stirring was continued for another 3 h at -78.degree. C. and then the reaction mixture was quenched with saturated aqueous NH.sub.4 Clsolution and the mixture allowed to warm to 25.degree. C. Water and CH.sub.2 Cl.sub.2 (250 mL each) were then added and the layers separated. The aqueous layer was extracted with CH.sub.2 Cl.sub.2 (2.times.250 mL) and the combined organic layers werewashed with brine (500 mL), dried (MgSO.sub.4) and concentrated in vacuo. The crude product was used directly in the next reaction without further purification.

Aldehyde 7011 as illustrated in FIG. 17. Ylide 7010 (1.15 equiv.) was added to a solution of aldehyde 7009 (1.0 equiv.) in CH.sub.2 Cl.sub.2 (0.5 M) and the mixture heated at reflux for 12 h. The mixture was then filtered through silica gelwashing with 2:1 ether:hexanes and the eluant reduced. The crude product was then purified by crystallization (hexanes) to afford aldehyde 7011 (82%).

Alcohol 7012. Aldehyde 7011 (1 equiv.) was dissolved in a mixture of anhydrous ether and CH.sub.2 Cl.sub.2 (1:1) [0.2 M] and the solution was cooled to -100.degree. C. (+)-Diisopinocampheylallyl borane (2.0 equiv. in pentane (prepared from(-)-Ipc.sub.2 BOMe (1.0 equiv.) and 1.0 equiv. of allyl magnesium bromide according to the method described previously.sup.1) was added dropwise under vigorous stirring, and the reaction mixture was allowed to stir for 1 h at the same temperature. Methanol was added at -100.degree. C., and the reaction mixture was allowed to warm up to room temperature. Amino ethanol (10.0 equiv.) was added and stirring was continued for 15 ether. The combined orgainc extracts were dried (MgSO.sub.4), filteredthe required acid chloride (1.1 equiv.). After stirring for 1 h, the reaction was quenched with saturated aqueous sodium bicarbonate solution. The layers were separated and the aqueous phase extracted with ether. The combined orgainc extracts weredried (MgSO.sub.4), filtered and concentrated in vacuo. Flash chromatography afforded alcohol 7012 (99%).